Ionotropic glutamate receptors comprise a large family of ligand-gated ion channels which are responsible for the majority of excitatory neurotransmission in the mammalian central nervous system. The subfamily of AMPA receptors mediates fast excitatory synaptic signaling on a millisecond timescale. In the continuous presence of glutamate, AMPA receptors desensitize rapidly and profoundly, thereby shaping the postsynaptic response at certain synapses. Mechanistic details on how the ion channel pore closes during desensitization with glutamate remaining bound to the receptor are missing and conclusive answers require high resolution structural information about the full-length receptor in the desensitized state. This application outlines the use of proven strategies to determine an atomic structure of a full-length AMPA receptor in complex with a high affinity agonist by X-ray crystallography. Preliminary results from this project include the successful crystallization of AMPA receptors in complex with a variety of different agonists and diffraction data from several crystal forms. The diffraction limit of these crystals needs to be improved by extensive optimization of the construct and crystallization conditions. The effects of various construct modifications on gating and desensitization will be monitored in electrophysiological experiments. Crystallization trials will not be limited to classical methods using detergent/protei micelles but also include new techniques which allow crystallization in a lipid-rich environment, such as bicelle and lipidic cubic phase crystallization. Furthermore, co-crystallization with FAB fragments from conformation-specific antibodies will be tested to reduce conformational heterogeneity in the crystal. Comparison of the new X-ray structural model of the AMPA receptor in the desensitized state to the known closed, non-desensitized state structure will reveal putative conformational changes underlying receptor desensitization. These predicted rearrangements will be tested by functional experiments to confirm the mechanistic concepts concluded from the structural findings. The expected insights from this application will not only improve our understanding of AMPA receptor desensitization on a molecular level, but also pave the way for designing novel therapeutic agents which enhance or reduce desensitization for the potential treatment of seizures, neurological and psychiatric disorders. PUBLIC HEALTH RELEVANCE: AMPA-type glutamate receptors are ligand-gated ion channels mediating fast excitatory postsynaptic currents in the brain. Drugs enhancing AMPA receptor desensitization, a poorly understood process resulting in channel closure in sustained presence of glutamate, are useful for the effective treatment of seizures, epilepsy and excitotoxicity-related diseases, like ischemic stroke. The structural insights from this applicatio will provide a molecular blueprint for novel designs of therapeutic compounds targeting AMPA receptor desensitization.